Bottom Line:
Most of these phenotypes are also found to depend on another TREX-2 subunit, Thp1.These results unveil a new role for Sem1 in the activation of the SAGA-dependent gene GAL1 and influencing H2B deubiquitylation.Our work provides insights into a novel functional relationship between Sem1 and the SAGA complex.

ABSTRACTTranscription and mRNA export are linked processes. However, the molecular mechanisms of this coordination are not clear. Sus1 (hENY2) participates in this coordination as part of two protein complexes: SAGA, a transcriptional co-activator; TREX-2, which functions in mRNA biogenesis and export. Here, we investigate the coordinated action of SAGA and TREX-2 required for gene expression. We demonstrate that TREX-2 subunit Sem1 also participates in transcription activation. Like Sus1, Sem1 is required for the induction of ARG1 and GAL1, these being SAGA-regulated genes. Chromatin immunoprecipitations show that proper recruitment of certain SAGA subunits to the GAL1 promoter depends on Sem1. Notably, both in vivo and in vitro analyses reveal that Sem1 influences SAGA-dependent histone H2B deubiquitylation. Most of these phenotypes are also found to depend on another TREX-2 subunit, Thp1. These results unveil a new role for Sem1 in the activation of the SAGA-dependent gene GAL1 and influencing H2B deubiquitylation. Our work provides insights into a novel functional relationship between Sem1 and the SAGA complex.

gkt272-F6: The TREX-2 subunit Thp1 influences SAGA–DUB-mediated activity. (A) Ubp8-TAP wild-type and thp1Δ cells were subjected to an in vitro deubiquitylation assay. The TAP purification of Ubp8 TAP-tagged cells was performed, and native calmoduline eluates were incubated with purified histone H2B. Sole purified histone H2B was incubated and used as a negative control. Error bars represent SE for at least three independent experiments. (B) The time course analysis of GAL1 induction in wild-type and thp1Δ cells for 120 min in the presence of 2% galactose. The relative GAL1 mRNA levels were normalized to SCR1. Values were referred to each strain at the zero time point. Error bars represent SE for at least three independent experiments. (C) The Ada2 (HAT/CORE) occupancy at the GAL1 promoter was monitored by ChIP analysis in wild-type and thp1Δ cells after 25 min of galactose induction. The occupancy level was calculated as the signal ratio of IP samples in relation to the input signal minus the background of a no-antibody control. The resulting normalized ratios were plotted. Levels of the Ada2-MYC bait protein were detected from the inputs by western blot using the anti-MYC antibodies (bottom). (D) The Taf9 (SA_TAF) occupancy at the GAL1 promoter was monitored by ChIP analysis in wild-type and thp1Δ cells after 25 min of galactose induction. The occupancy level was calculated as in C. The resulting normalized ratios were plotted. Levels of the Taf9-MYC bait protein were detected from the inputs as in C (bottom). (E) The Sus1 (DUB) occupancy at the GAL1 promoter was monitored and calculated as in C. The resulting normalized ratios were plotted. Levels of the Sus1-MYC bait protein were detected from the inputs as in C (bottom). Error bars represent SE for at least three independent experiments. All the qPCR primer sequences are provided in Supplementary Table S2.

Mentions:
Sem1 affects the recruitment of different SAGA subunits and enhances SAGA-mediated histone H2B deubiquitylation. Whether this is related to its role in stabilizing TREX-2 is unknown. To test whether the mutation in other TREX-2 subunit also lead to similar phenotypes, in vitro deubiquitylation activity, GAL1 induction and SAGA recruitment were investigated in a thp1Δ strain. As shown in Figure 6A, the absence of Thp1 leads to a significant reduction of Ubp8-mediated DUB activity, similarly to that observed for Sem1 deletion (Figure 5C). Moreover, as observed for sem1Δ, the absence of THP1 impairs GAL1 induction (Figure 6B). Finally, the ChIP experiments done to monitor recruitment of different SAGA factors to the GAL1 promoter in the wild-type and thp1Δ on the shift to galactose reveal that THP1 deletion decreased the correct recruitment of distinct SAGA subunits to the GAL1 promoter (Figure 6C and D). As it happens for sac3Δ (50), the presence of Sus1 is also compromised in the thp1Δ under the same conditions (Figure 6E). In conclusion, TREX-2 subunit Thp1 is needed for a precise SAGA recruitment to GAL1. All in all, the data herein suggest that Sem1 influences the GAL1 expression by a mechanism that interferes with SAGA-DUB-mediated activity and by stabilizing the TREX-2 complex.Figure 6.

gkt272-F6: The TREX-2 subunit Thp1 influences SAGA–DUB-mediated activity. (A) Ubp8-TAP wild-type and thp1Δ cells were subjected to an in vitro deubiquitylation assay. The TAP purification of Ubp8 TAP-tagged cells was performed, and native calmoduline eluates were incubated with purified histone H2B. Sole purified histone H2B was incubated and used as a negative control. Error bars represent SE for at least three independent experiments. (B) The time course analysis of GAL1 induction in wild-type and thp1Δ cells for 120 min in the presence of 2% galactose. The relative GAL1 mRNA levels were normalized to SCR1. Values were referred to each strain at the zero time point. Error bars represent SE for at least three independent experiments. (C) The Ada2 (HAT/CORE) occupancy at the GAL1 promoter was monitored by ChIP analysis in wild-type and thp1Δ cells after 25 min of galactose induction. The occupancy level was calculated as the signal ratio of IP samples in relation to the input signal minus the background of a no-antibody control. The resulting normalized ratios were plotted. Levels of the Ada2-MYC bait protein were detected from the inputs by western blot using the anti-MYC antibodies (bottom). (D) The Taf9 (SA_TAF) occupancy at the GAL1 promoter was monitored by ChIP analysis in wild-type and thp1Δ cells after 25 min of galactose induction. The occupancy level was calculated as in C. The resulting normalized ratios were plotted. Levels of the Taf9-MYC bait protein were detected from the inputs as in C (bottom). (E) The Sus1 (DUB) occupancy at the GAL1 promoter was monitored and calculated as in C. The resulting normalized ratios were plotted. Levels of the Sus1-MYC bait protein were detected from the inputs as in C (bottom). Error bars represent SE for at least three independent experiments. All the qPCR primer sequences are provided in Supplementary Table S2.

Mentions:
Sem1 affects the recruitment of different SAGA subunits and enhances SAGA-mediated histone H2B deubiquitylation. Whether this is related to its role in stabilizing TREX-2 is unknown. To test whether the mutation in other TREX-2 subunit also lead to similar phenotypes, in vitro deubiquitylation activity, GAL1 induction and SAGA recruitment were investigated in a thp1Δ strain. As shown in Figure 6A, the absence of Thp1 leads to a significant reduction of Ubp8-mediated DUB activity, similarly to that observed for Sem1 deletion (Figure 5C). Moreover, as observed for sem1Δ, the absence of THP1 impairs GAL1 induction (Figure 6B). Finally, the ChIP experiments done to monitor recruitment of different SAGA factors to the GAL1 promoter in the wild-type and thp1Δ on the shift to galactose reveal that THP1 deletion decreased the correct recruitment of distinct SAGA subunits to the GAL1 promoter (Figure 6C and D). As it happens for sac3Δ (50), the presence of Sus1 is also compromised in the thp1Δ under the same conditions (Figure 6E). In conclusion, TREX-2 subunit Thp1 is needed for a precise SAGA recruitment to GAL1. All in all, the data herein suggest that Sem1 influences the GAL1 expression by a mechanism that interferes with SAGA-DUB-mediated activity and by stabilizing the TREX-2 complex.Figure 6.

Bottom Line:
Most of these phenotypes are also found to depend on another TREX-2 subunit, Thp1.These results unveil a new role for Sem1 in the activation of the SAGA-dependent gene GAL1 and influencing H2B deubiquitylation.Our work provides insights into a novel functional relationship between Sem1 and the SAGA complex.

ABSTRACTTranscription and mRNA export are linked processes. However, the molecular mechanisms of this coordination are not clear. Sus1 (hENY2) participates in this coordination as part of two protein complexes: SAGA, a transcriptional co-activator; TREX-2, which functions in mRNA biogenesis and export. Here, we investigate the coordinated action of SAGA and TREX-2 required for gene expression. We demonstrate that TREX-2 subunit Sem1 also participates in transcription activation. Like Sus1, Sem1 is required for the induction of ARG1 and GAL1, these being SAGA-regulated genes. Chromatin immunoprecipitations show that proper recruitment of certain SAGA subunits to the GAL1 promoter depends on Sem1. Notably, both in vivo and in vitro analyses reveal that Sem1 influences SAGA-dependent histone H2B deubiquitylation. Most of these phenotypes are also found to depend on another TREX-2 subunit, Thp1. These results unveil a new role for Sem1 in the activation of the SAGA-dependent gene GAL1 and influencing H2B deubiquitylation. Our work provides insights into a novel functional relationship between Sem1 and the SAGA complex.